Taste transduction is initiated when chemical stimuli interact with the apical membrane of taste receptor cells. The interaction ultimately leads to an increase in intracellular calcium and release of transmitter from the taste cell onto gustatory nerve fibers. During the next funding period aspects of both transduction and transmitter release will be examined. The first two specific aims focus on sweet transduction, while the third aim examines transmitter release from taste cells. Aim 1: To determine the role of gustducin in sweet transduction. In the previous funding period evidence was provided that sugars activate the cAMP pathway, while synthetic sweeteners activate a pathway involving IP/3 and DAG. These two second messenger pathways apparently converge on a K+ conductance to depolarize taste cells. In the next funding period experiments are proposed to examine the role of the chemosensory-specific G protein, gustducin, in this process. A transgenic line of mice will be obtained from Dr. R Margolskee in which the gustducin promoter has been linked to the gene for Green Fluorescent Protein (GFP). Thus, taste cells that express gustducin will be fluorescent and can be targeted for physiological recording. Ca2+ imaging, whole cell recording, and human psychophysical studies will be used to determine the role of gustducin in sweet transduction. Aim 2: To identify the K+ channels that mediate the final step in sweet transduction. Single channel recording will be used to characterize the K+ channels that are closed in response to sweet stimuli. Specifically, experiments are proposed to determine whether the same channels are targeted by sugars and synthetic sweeteners, and whether phosphorylation is required for channel closure. Aim 3: To examine the role of IP/3 in neurotransmitter release from taste cells. Recent results on denatonium transduction in mudpuppy taste cells suggests that IP/3-mediate release of calcium from intracellular stores may be sufficient to trigger transmitter release from taste cells. In the next funding period synaptic vesicle release from taste cells will be imaged directly using the fluorescent membrane dye FM1-43. Experiments are proposed to determine the role of IP/3 and Ca/2+ in this process. These studies will provide important new information about the role of gustducin in sweet taste transduction and mechanisms involved in the release of transmitters from taste cells.